theoretical biology and medical modelling biomed central · 2.99 2.98 2.89 3.19 3.34 2.64 c20 c21...

BioMed Central

Theoretical Biology and Medical Modelling

ss
Open AcceResearchCommon angiotensin receptor blockers may directly modulate the immune system via VDR, PPAR and CCR2bTrevor G Marshall*1, Robert E Lee2 and Frances E Marshall3
Address: 1Autoimmunity Research Foundation, Thousand Oaks, California 91360, USA, 2Black Hawk College, Moline, Illinois 61443, USA and 3Los Robles Regional Medical Centre, Thousand Oaks, California 91360, USA

Email: Trevor G Marshall* - [email protected]; Robert E Lee - [email protected]; Frances E Marshall - [email protected]

* Corresponding author

AbstractBackground: There have been indications that common Angiotensin Receptor Blockers (ARBs)may be exerting anti-inflammatory actions by directly modulating the immune system. We decidedto use molecular modelling to rapidly assess which of the potential targets might justify the expenseof detailed laboratory validation. We first studied the VDR nuclear receptor, which is activated bythe secosteroid hormone 1,25-dihydroxyvitamin-D. This receptor mediates the expression ofregulators as ubiquitous as GnRH (Gonadatrophin hormone releasing hormone) and theParathyroid Hormone (PTH). Additionally we examined Peroxisome Proliferator-ActivatedReceptor Gamma (PPARgamma), which affects the function of phagocytic cells, and the C-CChemokine Receptor, type 2b, (CCR2b), which recruits monocytes to the site of inflammatoryimmune challenge.

Results: Telmisartan was predicted to strongly antagonize (Ki≈0.04nmol) the VDR. The ARBsOlmesartan, Irbesartan and Valsartan (Ki≈10 nmol) are likely to be useful VDR antagonists at typicalin-vivo concentrations. Candesartan (Ki≈30 nmol) and Losartan (Ki≈70 nmol) may also usefullyinhibit the VDR. Telmisartan is a strong modulator of PPARgamma (Ki≈0.3 nmol), while Losartan(Ki≈3 nmol), Irbesartan (Ki≈6 nmol), Olmesartan and Valsartan (Ki≈12 nmol) also seem likely tohave significant PPAR modulatory activity. Olmesartan andIrbesartan (Ki≈9 nmol) additionally actas antagonists of a theoretical modelof CCR2b. Initial validation of this CCR2b model wasperformed, and a proposed model for the AngiotensinII Type1 receptor (AT2R1) has beenpresented.

Conclusion: Molecular modeling has proven valuable to generate testable hypotheses concerningreceptor/ligand binding and is an important tool in drug design. ARBs were designed to act asantagonists for AT2R1, and it was not surprising to discover their affinity for the structurally similarCCR2b. However, this study also found evidence that ARBs modulate the activation of two keynuclear receptors-VDR and PPARgamma. If our simulations are confirmed by experiment, it ispossible that ARBs may become useful as potent anti-inflammatory agents, in addition to theircurrent indication as cardiovascular drugs.

Published: 10 January 2006

Theoretical Biology and Medical Modelling 2006, 3:1 doi:10.1186/1742-4682-3-1

Received: 07 December 2005Accepted: 10 January 2006

This article is available from: http://www.tbiomed.com/content/3/1/1

© 2006 Marshall et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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BackgroundWhy would ARBs have dose-dependent efficacy?Angiotensin Receptor Blockers (ARBs) act as antagonistsof the AngiotensinII Type1 receptor (AT2R1) [Swiss-Prot:P30556], and were designed to treat moderate hyper-tension. Although ARBs have been marketed for nearly adecade, their mode of action is not fully understood, anddebate still rages whether Angiotensin ConvertingEnzyme Inhibitors (ACEI) or ARBs are superior at reduc-ing ultimate mortality due to cardiovascular dysfunction.

An editorial in the New England Journal of Medicine con-cluded [1]:

"in two recently reported clinical trials in which the investiga-tors were allowed to increase the dose of Losartan gradually to100 mg per day, there was a significant reduction in the inci-dence of heart failure among high-risk patients; this findingraises the important question of whether higher doses of Losar-tan might have been more effective in reducing the rates of car-diovascular events"

Yet in-vitro studies [2] have shown that the ARBs producean efficient and total blockade of the Angiotensin II Type1 receptor (AT2R1) at doses much lower than this edito-rial was contemplating. There should be no dose relatedeffects once a total receptor blockade is place, so the obvi-ous question arises "how can an ARB have dose-depend-ent efficacy?"

It is accepted that diabetic nephropathy is beneficiallyaffected by ARBs [3-6], yet again the mechanisms, andoptimal dosage, remain elusive. A study using Irbesartannoted dosage-dependant efficacy, with significantlygreater protection at 300 mg/day versus 150 mg/day [4].

Schieffer, et.al. [7], found that ARBs appeared to exertstronger systemic anti-inflammatory and anti-aggregatoryeffects compared with ACEIs in Atherosclerosis. Luno,et.al. [8], recently reviewed studies which have shown thatACE Inhibitors (ACEI) did not always lead to the same

clinical outcome as ARBs, especially where the patient wassuffering from inflammatory diseases such as diabetes.

The reason for this is not immediately obvious, as ACE'sfunction is to cleave the octapeptide Angiotensin II fromAngiotensin I. The AngiotensinII then binds to AT2R1receptors on the activated phagocytes, an action inhibitedby the ARBs. Interrupting either pathway, with either ACEIor ARBs, should have the same effect – the activatedphagocytes will be denied Angiotensin II bound at theirreceptors.

Waterhouse, et.al. [9], and Marshall, et.al. [10], noted thatpatients with autoimmune disease were anecdotallyreporting that ARBs prescribed for hypertension caused anoticeable change in their perceived immune diseasesymptoms, a change not easily explained in terms ofhypertension, or hypotension, alone. We consequentlydecided to investigate whether molecular modellingcould help define precise mechanism(s) of action of theARBs upon inflammatory disease. Do they perhaps act asantagonists for receptors other than AT2R1? Immune sys-tem receptors, for example?

Identifying target nuclear and transmembrane receptors1. The VDRThe T-helper Type 1 (Th1) immune response is usuallydefined as one which generates significant quantities ofthe cytokine Interferon-gamma [11]. Many chronic dis-eases are associated with Th1 inflammation [12], includ-ing atherosclerosis [13], diabetes [14], and perhaps evenasthma [15].

Generation of Interferon-gamma in a Th1 activated mac-rophage catalyzes its mitochondrial production of thesecosteroid hormone 1,25-dihydroxyvitamin-D (1,25-D)by as much as 30-fold [16]. 1,25-D is the active secoster-oid of the Vitamin-D metabolism [9]. This steroid's pres-ence is often ignored by clinical medicine, since itcirculates in low concentrations (typically 75 picomoles/Litre, 29 pg/ml), which are very difficult to measure. Yet

Table 1: Estimated Inhibition Constant, Ki (nmol), for ARBs docking into several immune system receptors.

Olmesartan Telmisartan Valsartan Irbesartan Candesartan Losartan

VDR,1DB1 12, 27 0.038 14 10 35 77VDR,1TXI 10,34 0.039 14 12 30 74

PPAR 12 0.29 12 6 61 3CCR2b * 9* 25* 22* 9* 39* 25*AT2R1 * 0.10* 0.10* 0.3* 0.17* 1.5* 0.50*

*Note 1: CCR2b and AT2R1 are theoretical models, and may not be reliable (see text)Note 2: (conventional ligand binding data): 1,25-dihydroxyvitamin-D docks into VDR (PDB:1DB1) with Ki = 0.029 nmol and into VDR (PDB:1TXI) with Ki= 0.059 nmolTX522 docks into VDR (PDB:1DB1) with Ki = 0.071 nmol and VDR (PDB:1TXI) with Ki = 0.12 nmolTAK779 docks into putative CCR2b with Ki = 10 nmolGI262570 docks into PPAR (PDB:1FM9) with Ki = 0.040 nmol.


http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1DB1

http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1TXI



http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1FM9


1,25-D and its receptor, the Vitamin-D Receptor (VDR)[Swiss-Prot:P11473], are expressed in over 30 target tis-sues, and their expression is tightly coupled with regula-tors as ubiquitous as GnRH (Gonadatrophin hormonereleasing hormone) [17], and the Parathyroid Hor-mone(PTH) [18].

Ripple-down effects of VDR activation include changesnot only to the androgens and thyroid hormones, but alsoto ACTH, Insulin Receptors, P450C1, and many other bio-logically important metabolites [18,46].

In patients with severe Th1 immune disease, clinicalobservations [9,10] indicated that the administration ofthe ARB Olmesartan, at a concentration in excess of thatneeded for full AT2R1 antagonism, often causes the levelof circulating 1,25-D to drop.

We therefore decided to target the VDR nuclear receptor[19] for further study.

2. Peroxisome Proliferator Activated Receptors (PPARs)Benson, et.al. reported [20] that the ARB 'Telmisartan'seems to act both as an agonist and antagonist of Peroxi-some Proliferator Activated Receptor gamma (PPAR-gamma) [Swiss-Prot:P37231], a nuclear hormonereceptor from the same 'NR1' subfamily as VDR. ThePPARs act as anti-inflammatory transcription factors [21].Part of this anti-inflammatory regulation is mediatedthrough negative interference between PPARs and nuclear

VDR binding pocket showing primary 1,25-D docking resi-duesFigure 4VDR binding pocket showing primary 1,25-D docking residues. Note: 1,25-D depicted with yellow backbone for visual clarity. Carbon atoms shown as grey, oxygen as red, nitrogen as blue, polar hydrogen as blue-white. Non-polar hydrogens not displayed. Residues displayed as 'CPK' charge spheres, ligand in 'ball and stick' format.

VDR-docked configurations for 1,25-D and Telmisartan, sep-arately and superimposedFigure 2VDR-docked configurations for 1,25-D and Telmisar-tan, separately and superimposed. Note: Models depicted as "thick" and "thin" solely for visual clarity. Carbon atoms shown as grey, oxygen as red, nitrogen shown as blue, polar hydrogen as blue-white. Non-polar hydrogens not dis-played.

1,25-D and TX522 with superimposed X-ray and VDR-docked configurationsFigure 11,25-D and TX522 with superimposed X-ray and VDR-docked configurations. Note: Carbon atoms shown as grey, oxygen as red. Hydrogens not displayed.

VDR-docked configurations for 1,25-D and Olmesartan, with superimposition showing both conformationsFigure 3VDR-docked configurations for 1,25-D and Olme-sartan, with superimposition showing both confor-mations. Note: Models depicted as "thick" and "thin" solely for visual clarity. Carbon atoms shown as grey, oxygen shown as red, nitrogen as blue, polar hydrogen as blue-white. Non-polar hydrogens not displayed.




2D LigPlot of 1,25-D bound into the VDR ligand binding pocketFigure 52D LigPlot of 1,25-D bound into the VDR ligand binding pocket. Note: The core structure of the hydrogen-bonded residues is expanded to a 'ball-and-stick' format, so as to show the atoms involved in hydrogen bond formation.

Key

Ligand bond

Non-ligand bond

3.0 Hydrogen bond & length

His 53 Non-ligand residues involved in hydrophobic

contact

Atoms involved in hydrophobic contact

2.99

2.98

2.89

3.19

3.34

2.64

C20

C21

C17

C22

C13

C16 C12

C14

C18

C15 C11

C8 C9

C7

C6

C5

C4

C10

C3

C1

C19

C2

O3

O1

C23 C24

C25

C26

C27 O25

N

CA

C

CB O

CG

CD1 CD2

CE1 CE2

CZ

OH

N

CA

C

CB

O

CG

CD

NE

CZ

NH1

NH2

N

CA

C

CB

O

CG

ND1

CD2

CE1

NE2

N CA

C

CB

O

OG

N

CA

C

CB

O

OG

N

CA

C

CB

O

CG

ND1

CD2

CE1

NE2

Trp 286

Ser 275

Tyr 295

Val 234

Phe 422

Leu 230

Ile 271

Cys 288

Val 300

Leu 313

1,25-D

Tyr 143

Arg 274

His 397

Ser 278

Ser 237

His 305



The VDR agonist TX522 in the VDR ligand binding pocketFigure 6The VDR agonist TX522 in the VDR ligand binding pocket. Note: The core structure of the hydrogen-bonded resi-dues is expanded to a 'ball-and-stick' format, so as to show the atoms involved in hydrogen bond formation.

Key

Ligand bond

Non-ligand bond



contact


3.02

2.88

2.80

3.22

2.71

C20

C21

C17

C22

C13

C16

C12

C14

C18 C15

C11

C8 C9

C7

C6

C5

C4

C10

C3

C1

C2

O3

O1

C23 C24

C27

C26

C25 O25

N

CA C

CB

O

CG CD1

CD2 CE1

CE2 CZ

OH

N

CA

C

CB

O

CG

ND1

CD2

CE1

NE2

N

CA

C

CB

O

CG

CD

NE

CZ

NH1 NH2

N

CA

C

CB

O

OG

N

CA

C

CB

O

CG

ND1

CD2

CE1 NE2

Ser 275

Trp 286

Tyr 295

Val 234

Ser 237

Phe 422

Leu 230

Cys 288

Val 300

Leu 313

TX522

Tyr 143

His 397

Arg 274

Ser 278

His 305



Olmesartan bound into the sterol terminus of the VDR binding pocketFigure 7Olmesartan bound into the sterol terminus of the VDR binding pocket. Note: This is the 12 nanomolar conforma-tion of Olmesartan in the binding pocket. The core structure of the hydrogen-bonded residues is expanded to a 'ball-and-stick' format, so as to show the atoms involved in hydrogen bond formation.

Key

Ligand bond

Non-ligand bond



contact


3.35

C1

C2 C6

C18

C3

C4

C5

C7

N1

C8

C9

N2

C10

C13

C14

C11

C12

C15

O1

O2

C16

C17

O3

C19 C20

C21 C23

C24

C22

N3

N4

N5

N6

N CA

C

CB

O

CG

CD NE

CZ

NH1

NH2Ser 275

Tyr 143

Val 300

Tyr 295Ser 237Leu 230

Leu 233

Val 234

Trp 286

Ile 271

His 305

Ser 278

Leu 313

Olmesartan

Arg 274



Telmisartan docked into the VDR ligand binding pocketFigure 8Telmisartan docked into the VDR ligand binding pocket. Note: Telmisartan is a strong antagonist of the VDR's activa-tion.

Key

Ligand bond

Non-ligand bond



contact


3.18

3.29

2.52

2.97

C1

C2

C3

C26

C4

C6

C5

C7

N1

N2

C8

C9

C23

C10

C11

C12

C13

C15

C14

C16

C17

C18

C19

C22

C21

C20

O1

O2

C24

C25

N3

N4

C27

C28

C30

C29

C31

C33

C32

N CA

C

CB

O

CG1

CG2

CD1

N

CA

C

CB

O

CG CD

NE

CZ

NH1

NH2

N

CA

C

CB

O

CG

ND1

CD2

CE1

NE2

N

CA

C

CB

O

OG

Ser 275

Leu 233

Tyr 143

Ala 231

Met 272

Leu 313Trp 286

Tyr 147

Phe 150

His 305

Leu 227 Leu 230

Cys 288

Leu 309

Leu 414

Val 418

Val 234

Val 300

Tyr 401

Telmisartan

Ile 271

Arg 274

His 397

Ser 237



Irbesartan docked into the VDR ligand binding pocketFigure 9Irbesartan docked into the VDR ligand binding pocket. Note: The core structure of the hydrogen-bonded residues is expanded to a 'ball-and-stick' format, so as to show the atoms involved in hydrogen bond formation.

Key

Ligand bond

Non-ligand bond



contact


3.23

C1

C2

C6

C19

C3

C4

C5

C7

N1 C8

C9 N2

C10

C14 O1

C11

C12 C13

C15

C16

C17

C18

C20

C21

C22

C24

C25

C23

N3

N4 N5

N6

N

CA C

CB

O

CG

CD

NE

CZ

NH1 NH2

Trp 286

Ser 237

Ile 271

Ser 275

Tyr 143

Tyr 295

Met 272

Leu 233

Leu 230

Tyr 147

Phe 150

Val 300

Ser 278

Cys 288

Irbesartan

Arg 274



Valsartan docked into the VDR ligand binding pocketFigure 10Valsartan docked into the VDR ligand binding pocket.

Key

Ligand bond

Non-ligand bond



contact


C1

C2

C6

C18

C3 C4

C5

C7

N1 C8

C13

O1

C9

C10

C11

C12

C14 C15 O2

O3

C16

C17

C19

C20 C21

C23

C24

C22

N2

N3

N4

N5

Ile 271

Arg 274

Ser 275

Leu 233

Tyr 143

Tyr 295

Ser 237

Ile 268

Ser 278

Trp 286

Leu 230

Met 272

Val 300

Tyr 147

Cys 288

Asp 299

Leu 313

Valsartan



Candesartan docked into the VDR ligand binding pocketFigure 11Candesartan docked into the VDR ligand binding pocket. Note: The core structure of the hydrogen-bonded residues is expanded to a 'ball-and-stick' format, so as to show the atoms involved in hydrogen bond formation.

Key

Ligand bond

Non-ligand bond



contact


3.07

3.18

C1

C2

C6

C7

C3

C4

C5

C18

N1

C8

C9

C10 C11

N2

O1

C12

C17

C14

C13

C15

C16

O2

O3

C19

C20

C21

C23

C24

C22 N3

N4

N5

N6

N

CA

C

CB

O

CG

CD

NE

CZ

NH1

NH2

N

CA

C

CB

O

SG

Trp 286

Tyr 295

Ser 237

Leu 233

Tyr 143

Leu 230

Met 272Val 234

Ser 278

Tyr 147

Ile 268

His 305

Ile 271

Leu 313

Ser 275

Candesartan

Arg 274

Cys 288



Losartan docked into the VDR ligand binding pocketFigure 12Losartan docked into the VDR ligand binding pocket. Note: The core structure of the hydrogen-bonded residues is expanded to a 'ball-and-stick' format, so as to show the atoms involved in hydrogen bond formation.

Key

Ligand bond

Non-ligand bond



contact


2.72 C1

C2 C6

C7

C3

C4

C5

C16

N1

C8

C9

C10

C11

N2

C12

CL1

O1

C13

C14

C15

C17

C18

C19

C20

C22

C21

N3

N4

N5

N6

N

CA

C

CB

O

OG

Ile 271

Tyr 143

Leu 233

Val 234

Met 272Leu 230

Trp 286

Ile 268

Ser 278Tyr 295

Arg 274

Ser 275

Phe 150

Tyr 236

Cys 288Tyr 147

Losartan

Ser 237


factors such as NF-kappaB. Ligands of PPAR may affect theinflammatory response in diseases as wide-ranging asInflammatory Bowel Diseases, Atherosclerosis, Parkin-son's Disease and Alzheimer's [22]. Clearly, it is impor-tant to know exactly how the ARBs might affectPPARgamma.

3. C-C chemokine receptor type 2 (CCR2b)Monocyte chemotactic protein-1 (MCP-1) binding to itsreceptor, CCR2b [EMBL:BC095540], plays an importantrole in a variety of diseases involving infection, inflamma-tion, and/or injury [23,24]. CCR2b recruits monocytes tothe sites of tissue damage. The monocytes later differenti-ate to macrophages and/or polymorphonucleated 'giant'cells.

CCR2b belongs to the same family of 7-TransmembraneG-Protein Coupled Receptors (GPCRs) [25] as doesAT2R1, and the similarities between these two GPCRs,together with the clinical observations [9,10], supportedthe addition of CCR2b to this study.

ResultsValidation of 'AutoDock' simulation softwareIt was decided to use automated docking of the ligands soas to minimize subjective factors which might arise if theligands were fitted into the binding pockets manually. TheScripps' package, AutoDock [26-28], was selected for thistask. Toprakci, et.al. [29], recently compared the Ki valuesestimated by AutoDock for ten inhibitors of humanmonoamine oxidase-B, with the values of Ki which hadbeen determined by experiment. In every case, there wasless than one order of magnitude difference between theexperimentally determined Ki, and the value estimated bycomputer simulation of the ligand-bound enzyme. Chen,et.al. [30], also concluded that AutoDock provided accu-rate estimation of ligand-DNA binding parameters.

We were able to compare calculated Ki for some of ourdocking experiments with published values, and similarlyfound excellent agreement. For example, we validated ourPPARgamma model by docking the ligand GI262570(Farglitazar), essentially as predicted by the data of Xu,et.al. [31].

Table 2: Multiple sequence alignment for AT2R1 and Bovine Rhodopsin (PDB:1L9H)

sp|P30556|AGTR1_HUMANgi|21465997|pdb|1L9H|A








SeqA Name

1sp|P30556|AGTR1_HUMAN

---------MILNSSTEDGIKRIQDDCPKAGRHN-YIFVMIPTLYSIIFV 40XMNGTEGPNFYVPFSNKTGVVRSPFEAPQYYLAEPWQFSMLAAYMFLLIM 50

: : * . : *: * : . * : : : * * : . : : : : :

VGIFGNSLVVIVIYFYMKLKTVASVFLLNLALADLCFLLTLPLWAVYTAM 90LGFPINFLTLYVTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSL 100

: * : * * . : * : ** :* . :***** : * ** : : : : :** : :

EYRWPFGNYLCKIASASVSFNLYASVFLLTCLSIDRYLAIVHPMKSRLRR 140HGYFVFGPTGCNLEGFFATLGGEIALWSLVVLAIERYVVVCKPMSN-FRF 149 . : ** * : : . . : : . : : : * . * : *:** : . : :** . . : *

TMLVAKVTCIIIWLLAGLASLPAIIHRNVFFIENTNITVCAFHYESQNST 190GENHAIMGVAFTWVMALACAAPPLVGWSRYIPEGMQCSCGIDYYTPHEET 199

* : : * : : * . : * . : : . : : * . : : : * . : : . *

LPIGLGLTKNILGFLFPFLIILTSYTLIWKALKKAYEIQKN----KPRND 236NNESFVIYMFVVHFIIPLIVIFFCYGQLVFTVKEAAAQQQESATTQKAEK 249 . : : : : *: :* : : : *: . * : : : * : * * : : : : .

DIFKIIMAIVLFFFFSWIPHQIFTFLDVLIQLGIIRDCRIADIVDTAMPI 286EVTRMVIIMVIAFLICWLPYAGVAFYIFTHQG--------SDFGPIFMTI 291 : : : : : : : * : *: : . * : * : . : * . * . . . : * : * . *

TICIAYFNNCLNPLFYGFLGKKFKRYFLQLLKYIPPKAKSHSNLSTKMST 336PAFFAKTSAVYNPVIYIMMNKQFRNCMVTTLCCG----KNPLGDDEASTT 337. :* . ** : :* : : . * : * : . : : * ...:* . . . : *

LSYRPSDNVSSSTKKPAPCFEVE 359V S K T E T S Q V A P A - - - - - - - - - - - 349: * : . : * : . : : . . . : . . . .

Len(aa) SeqB Name Len(aa) Score

359 2 gi|21465997|pdb|1L9H|A 349 17


http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1L9H


It is important to understand that the 'Lamarckian geneticalgorithm' used by AutoDock does not guarantee conver-gence to an optimal solution. The existence of the 'opti-mal' solution, amongst any set of docking results, onlybecomes assured as the number of docking attempts tendsto infinity. Considerable computing power was expendedin order to maximize the likelihood that this study identi-fied the lowest energy docking configurations. Addition-ally, the algorithm's convergence parameters weremanually adjusted whenever successive docking runs werenot returning consistent minima.

ARBs exhibit a strong affinity for VDR ligand binding pocketIn order to maximize reliability, two discrete models wereused for the ligand binding pocket of the VDR, extractedfrom two separate X-ray generated structures. The firstmodel was "The crystal structure of the nuclear receptorfor vitamin D bound to its natural ligand" [32][PDB:1DB1], while the second was the VDR bound to theagonist TX522 [33] [PDB:v].

There was no significant difference between the resultsobtained from either VDR structure. Table 1 shows the

predicted inhibition constants (Ki), in nanomoles, foreach of the ARBs binding into [PDB:1DB1] and[PDB:1TXI].

As a further check of model validity, 1,25-D was initiallydocked into [PDB:1DB1] with a Ki = 0.03 nmol and into[PDB:1TXI] with Ki = 0.06 nmol. TX522 was then dockedinto [PDB:1DB1] with Ki = 0.07 nmol and [PDB:1TXI]with Ki = 0.12 nmol. The difference between the crystalstructure of the ligands and the predicted docked confor-mations was very small (Figure 1), and seems primarilydue to AutoDock's reliance upon grid-based energy calcu-lations.

The ARB 'Telmisartan' had a strong affinity for the VDR,with Ki≈0.04 nmol into either structure. This value is closeto that achieved by 1,25-D itself, which yielded Ki≈0.03nmol into [PDB:1DB1] and Ki≈0.09 nmol into[PDB:1TXI]. Telmisartan docked with a conformationuncannily similar to 1,25-D (see Figure 2).

Irbesartan and Valsartan gave predicted Ki values in the10–14 nanomolar region, probably indicating significant

Table 3: Multiple sequence alignment for CCR2b and Bovine Rhodopsin (PDB:1L9H)

1kp1_A (CCR2b)gi|21465997|pdb|1L9H|A








SeqA Name

2gi|21465997|pdb|1L9H|A

MLSTSRSR--FIRNTNESGEEVTTFFDYDYGAPCHKFDVKQIGAQLLPPL 48XMNGTEGPNFYVPFSNKTGVVRSPFEAPQY------YLAEPWQFSMLAAY 44 : . : . . : : : * : : * : .* : *.:.. . : . : . : * . .

YSLVFIFGFVGNMLVVLILINCKKLKCLTDIYLLNLAISDLLFLIT--LP 96MFLLIMLGFPINFLTLYVTVQHKKLRTPLNYILLNLAVADLFMVFGGFTT 94 * : : : : * * * : *. : : : : * * *: : ***** : : **: : : : .

LWAHSAANEWVFGNAMCKLFTGLYHIGYFGGIFFIILLTIDRYLAIVHAV 146TLYTSLHGYFVFGPTGCNLEGFFATLGGEIALWSLVVLAIERYVVVCKPM 144 * . : *** : * : * : : * . : : : : : * :* : ** : . : : . :

FALKARTVTFGVVTSVITWLVAVFASVPGII-FTKCQKEDSVYVCGP--Y 193SNFRFG-ENHAIMGVAFTWVMALACAAPPLVGWSRYIPEGMQCSCGIDYY 193 : : : . . . : : . :** : : * : . : . * : : : : : * . ** *

FPRGWNN--FHTIMRNILGLVLPLLIMVICYSGILKTLLRCRNEKKRHRA 241TPHEETNNESFVIYMFVVHFIIPLIVIFFCYGQLVFTVKEAAAQQQESAT 243 * : . * . . * : : : : : **: : : . : ** . : : * : . . : : : . :

VRVIFTIMIVYFLFWTPYNIVILLNTFQEFFGLSNCESTSQLDQATQVTE 291TQKAEKEVTRMVIIMVIAFLICWLPYAGVAFYIFTHQGSDFGPIFMTIPA 293. : . : . : : . : : * * : . : . : . : .

TLGMTHCCINPIIYAFVGEKFRRYLSVFFRKHITKRFCKQCPVFYRETVD 341FFAKTSAVYNPVIYIMMNKQFR-------NCMVTTLCCGKNPLGDDEAST 336 : . * . * * :** : : . : : ** : . : * . * : * : * :

GVTSTNTPSTGEQEVSAGL 360TVSKTETSQVAPA--- - - - 349 * : . * : * . . . . . : : .


349 3 1kp1_A 360 17














antagonistic action at concentrations safely achievable in-vivo.

Olmesartan similarly predicted useful Ki values, rangingfrom 10 to 34 nmol. Particularly interesting is that twodistinct conformations were identified.

Figure 3 shows that Olmesartan docked in each conforma-tion, one with its imidazole terminus near the triol of1,25-D. The second focused on the seco terminus of 1,25-D.

Losartan docked with a Ki around 70 nanomolar, Cande-sartan around 30 nanomolar. These are likely also signifi-cant antagonists, but higher dosage levels would benecessary.

Hydrogen bonds and hydrophobic contacts during docking with the VDRFigure 4 shows the ligand binding pocket of the VDR with1,25-D docked into it, highlighting those residues withwhich 1,25-D forms hydrogen-bonds.

Figure 5 is a 2D representation of the 3D structure of Fig-ure 4, created with Ligplot [53,54]. The hydrogen bondswere identified with HBPLUS [55,56], as were the hydro-phobic contacts formed between 1,25-D and the VDR res-idues. The core structure of the hydrogen-bonded residuesis expanded to a 'ball-and-stick' format so as to showwhich atoms are involved in hydrogen bond formation.

A double hydrogen bond was formed from the oxygen ofthe triol group of 1,25-D, both to the imidazole nitrogenof HIS305, and to the imidazole nitrogen of HIS397.Another hydrogen bond extends from the 1-hydroxyl oxy-gen to the aminoacetal of ARG274 and the hydroxyl ofSER237, and another pair from the ligand's O3 oxygen toSER278 and TYR143.

Figure 6 shows that the VDR agonist TX522 [42] alsoforms a double hydrogen bond between the oxygen of itstriol group, the imidazole of HIS397, and the imidazoleof HIS305. The 3-hydroxyl-oxygen is hydrogen-bonded toTYR 143 and SER278, while the 1-hydroxyl-oxygen formsa hydrogen bond with the aminoacetal of ARG274. Nohydrogen bond is formed with SER237, presumably due

Table 4: Multiple sequence alignment for AT2R1 and CCR2b

gi|4757938|ref|NP_000639.1|CCR2bgi|231519|sp|P30556|AGTR1_HUMA



gi|4757938|ref|NP_000639.1|gi|231519|sp|P30556|AGTR1_HUMA





SeqB Name

1gi|4757938|ref|NP_000639.1| 360

-MLSTSRSRFIRNTNESGEEVTTFFDYDYGAPCHKFDVK QIGAQLLPPLY 49MILNSSTEDGIKRIQDD---------------CPKAGRHNYIFVMIPTLY 35 : * . :* . * : . : : . . . . : : . . . : . * * . : : : :* . **

SLVFIFGFVGNMLVVLILINCKKLKCLTDIYLLNLAISDLLFLITLPLWA 99SIIFVVGIFGNSLVVIVIYFYMKLKTVASVFLLNLALADLCFLLTLPLWA 85* : : * : . * : . * * * * * : : : * * * : : . : : * * * * * : : * * * * : * * * * * *

HSAANE--WVFGNAMCKLFTGLYHIGYFGGIFFIILLTIDRYLAIVHAVF 147VYTAMEYRWPFGNYLCKIASASVSFNLYASVFLLTCLSIDRYLAIVHPMK 135 : * * * * * * : * *: : . : . : . . : * : : *:********* . :

ALKARTVTFGVVTSVITWLVAVFASVPGIIFTKCQKED--SVYVCGPYFP 195 SRLRRTMLVAKVTCIIIWLLAGLASLPAIIHRNVFFIENTNITVCAFHYE 185 : ** : . . ** . : * * * :* : ** : * .** . : : . : * * . : :

RGWNNFHT---IMRNILGLVLPLLIMVICYSGILKTLLRCRNEKKR---- 238SQNSTLPIGLGLTKNILGFLFPFLIILTSYTLIWKALKKAYEIQKNKPRN 235 . . : : : *** *: : : *:**: : .* : * * : * : . : : * .

HRAVRVIFTIMIVYFLFWTPYNIVILLNTFQEFFGLSNCESTSQLDQATQ 288DDIFKIIMAIVLFFFFSWIPHQIFTFLDVLIQLGIIRDCRIADIVDTAMP 285 . . : :* : : *: : . : * : * * : : *. :* : . : : : : : * . : . : * *

VTETLGMTHCCINPIIYAFVGEKFRRYLSVFFRKHITKRFCKQCPVFYRE 338ITICIAYFNNCLNPLFYGFLGKKFKRYFLQLLKYIPPKAKSHSNLSTKMS 335:* : . : * : * * : : * . * : * : * * : * * : : : : .* . : . .

TVDGVTSTNTPSTGEQEVSAGL-- 360TLSYRPSDNVSSSTKKPAPCFEVE 359* : . . * * . .* : : : . . .


360 2 gi|231519|sp|p30556|AGTR1_HUMA 359 27



to a lowered affinity consequent upon the removal of theC19 position carbon from 1,25-D(cf.Figure 4).

The Ki = 12E-9 configuration of Olmesartan (Figure 7),forms a hydrogen bond from its imidazole terminalhydroxyl to ARG274. Olmesartan forms only hydropho-bic contacts with the key VDR binding residues TYR143,SER237, SER278 and HIS305. TYR143 is especially impor-tant. It is part of the 'hinge region,' and key for VDR tran-scriptional activity [51,57]. It is thus almost certain thatOlmesartan will function as a VDR antagonist.

Telmisartan docks with a Ki of 0.04 nmol, so that typicalin-vivo concentrations of the ARB should be sufficient todisplace 1,25-D from the ligand binding domain. Figure 8shows that that hydrogen bonds are formed to SER237,ARG274, HIS397 and ILE271, but not to TYR143. SER278or HIS305. Telmisartan would thus seem likely to act as avery strong antagonist of the VDR, with an affinity signif-icantly stronger than the other ARBs.

Irbesartan (Figure 9) formed a hydrogen bond between itstetrazole group and the amino of ARG274. The lack ofhydrogen bonds to TYR143 and SER278 indicate thatIrbesartan will be a VDR antagonist.

Valsartan, although it exhibits a potentially useful affinityas a VDR antagonist, failed to form hydrogen bonds withany key residue (Figure 10).

The imidazole of Candesartan formed a bond with thesulphur of CYS288 (Figure 11), and the imidazole termi-nus oxygen of Losartan hydrogen-bonded withSER237(Figure 12). Both are indicative of actions antago-nistic to VDR activation.

ARBs exhibit an affinity for PPARgammaWe extracted the coordinate data for PPARgamma from[PDB:1FM9], an X-ray structure. As model validation, thePPARgamma agonist GI262570 (Farglitazar) was dockedwith Ki≈0.04 nmol, close to the (approx.) 0.01 nmol pre-dicted by the inhibition curve in figure 1A of Xu, et.al.[31].

Table 1 shows that the ARBs exhibited a strong affinity forthe ligand binding pocket of PPARgamma, with Ki rang-ing from 0.29 to 61 nanomoles.

Telmisartan is the strongest modulator of PPARgamma(Ki≈0.3 nmol), while Losartan (Ki≈3 nmol), Olmesartan(Ki≈12 nmol), Irbesartan (Ki≈6 nmol) and Valsartan(Ki≈12 nmol) also seem likely to have significant PPARmodulatory activity. Candesartan (Ki≈ 61 nmol) may alsohave useful activity at a higher dosage.

ARBs exhibit a strong affinity for CCR2bThe ARBs are designed as antagonists for the AngiotensinII Type 1 Receptor (AT2R1). This is a GPCR [36] of the"Class A (Rhodopsin-like) 7-transmembrane receptors."CCR2b is another Class A GPCR, with surprising similar-ity to AT2R1.

Table 2 shows the multiple sequence alignment betweenAT2R1 and Bovine Rhodopsin [PDB:1L9H], the prototypestructure for Class A GPCRs. Table 3 shows an alignmentfor CCR2b vs. Rhodopsin, while Table 4 compares AT2R1and CCR2b. It is interesting to note that CCR2b and

Overview of the ligand binding pocket identified in CCR2b (PDB:1KP1)Figure 13Overview of the ligand binding pocket identified in CCR2b (PDB:1KP1). Olmesartan is shown docked into pocket.


http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1KP1





AT2R1 both exhibit only 17% homology with BovineRhodopsin, while the score between them is much higher,at 27%.

There are no complete X-ray or NMR structures of Homosapiens' Class A GPCRs in PDB, or any other public data-base. However, Shi, et.al. [37] had derived a theoretical

Perspective view showing how pocket is located underneath Extracellular 'loop' 1. Olmesartan is shown docked into pocketFigure 14Perspective view showing how pocket is located underneath Extracellular 'loop' 1. Olmesartan is shown docked into pocket. Note: Residues displayed as 'CPK' charge spheres. Ligand displayed as stick and ball model. Left is view from front of pocket, facing helices 7 and 1, right view is from the top, looking across the top of helices 1 and 2.

CCR2b residues highlighted alongside docked TAK779. From left: front of pocket, rear of pocketFigure 15CCR2b residues highlighted alongside docked TAK779. From left: front of pocket, rear of pocket. Note: Carbon atoms shown as grey, oxygen as red, nitrogen as blue, polar hydrogen as blue-white, sulphur as yellow. Non-polar hydrogens not displayed. Residues displayed as 'CPK' charge spheres, ligand as 'ball and stick' models.




TAK779 docked into the CCR2b binding pocketFigure 16TAK779 docked into the CCR2b binding pocket.

Key

Ligand bond

Non-ligand bond



contact


C1

N1

O1

C2

C20

C3

C4

C5

C19

C6

C7

C8

C9

C11

C10

C12

C13 C14

C15 C18

C16

C17

C21

C22 C23

C25 C24

C26 N2

C27

C28

C29

C30 C31

C32 C33

O2

Leu 45

Thr 292

Gly 41

Tyr 188

Ser 186

Val 37

Thr 296

Ile 300

His 297

Pro 31Cys 32

Ile 304

Leu 44

Asn 301

His 33

Leu 293

Tak779


model, [PDB:1KP1], which provided a basis for us tostudy. We tried to improve upon [PDB:1KP1] by using,inter alia, Truncated Newton energy minimization withPonder's TINKER Tools [38,39] and homology modellingwith Sali's 'Modeller' [40,41]. However, even extensivehomology modelling against the Bovine Rhodopsin X-raystructure [PDB:1L9H], and other theoretical models, suchas [PDB:1KPX], failed to improve upon [PDB:1KP1].

We accepted that [PDB:1KP1] was probably a valid modelfor CCR2b based on the detailed nature of Shi, et.al's stud-ies [37], our failed attempts to improve upon it, and themanner in which it docked, exactly as predicted, with theCCR2b antagonist, TAK779.

A binding pocket exists between helices seven and one of[PDB:1KP1], extending back to extracellular regions oneand three. Baba, et.al. [42] had measured the inhibitoryeffects of Tak779 on CCR2b in their laboratory, showingan experimental Ki≈9 nmol. When we docked TAK779into our putative binding pocket, it predicted a Ki≈10nmol, essentially identical with this experimental value.

Figure 13 shows the location of this binding pocket, andFigure 14 an overview of the pocket structure, runningbetween GPCR helices seven and one, beneath the extra-cellular regionone, and bounded at the rear by extracellu-lar region three.

Figure 15 shows the residues binding TAK779 into theputative pocket. Hydrophobic interactions with LEU45,HIS297, ILE300, TYR188, PRO31 and CYS32, help to sta-bilize the ligand. The 2D LigPlot of residue interactionscan be seen at Figure 16.

Olmesartan and Irbesartan each showed excellent affinity(Ki≈9 nmol) for this binding pocket, while Valsartan, Tel-misartan, Candesartan and Losartan exhibited slightly less(Kifrom 22 to 40 nmol).

Figure 17 shows the residues which interact with Olme-sartan. A hydrogen bond is formed with the imidazole ofHIS297, while ILE300, ALA42, LEU45, THR292, TYR188,CYS32 and PRO31 all help to stabilize the ligand. Figure18 shows the 2D LigPlot of these interactions.

Figure 19 shows the docked position of TAK779 and Olm-esartan superimposed, to enable easier comparison of thefinal location of each ligand.

Irbesartan forms hydrophobic contacts with a set of resi-dues similar to that of Olmesartan (see Figure 20).

The ARBs, and TAK779, not only fill space within thisbinding pocket, but also 'anchor' the top of helices sevenand one to extracellular regions three and one, restrainingthe motion of GPCR elements, and, most probably, inhib-iting its activation [43].

A putative AT2R1 receptor modelA primary goal set for this study had been the validationof every structure and tool we used. It had therefore beendecided to ensure that the ARBs would dock into AT2R1with inhibition constants close to the values measured in-vitro, as documented in the various FDA New Drug Appli-cations (NDAs). For example, NDA21-286 [2], indicates aKi for Olmesartan and Candesartan of approx. 0.1nanomolar, and for Losartan about 3 times higher.

This validation task proved to be the most difficult of thestudy. There was no AT2R1 X-ray structure publicly avail-able, nor any comprehensive theoretical model. Addition-ally, there was very little comparative experimental ARBdata available (FDA NDA21-286 is the exception to this).Most authors studied only one commercial ARB productin isolation.

We tried to use the theoretical model published by Mar-tin, et.al. [43] [PDB:1ZV0] for an activated AT2R1. But noARB would bind to that receptor configuration, even afterthe extensive energy optimization required to move helixseven back into its un-activated position.

CCR2b residues highlighted alongside docked Olmesartan, viewed from the front of the binding pocketFigure 17CCR2b residues highlighted alongside docked Olme-sartan, viewed from the front of the binding pocket. Note: Carbon atoms shown as grey, oxygen as red, nitrogen as blue, polar hydrogen as blue-white, sulphur as yellow. Non-polar hydrogens not displayed. Residues displayed as 'CPK' charge spheres, ligand as 'ball and stick' models.


http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1BFM

http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1BFM







http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1KPX




http://www.rcsb.org/pdb/cgi/explore.cgi?pdbId=1ZV0



Olmesartan docked into the CCR2b binding pocketFigure 18Olmesartan docked into the CCR2b binding pocket. Note: The core structure of the hydrogen-bonded residues is expanded to a 'ball-and-stick' format, so as to show the atoms involved in hydrogen bond formation.

Key

Ligand bond

Non-ligand bond



contact


3.29

C1

C2

C6

C18

C3 C4

C5

C7

N1 C8

C9 N2

C10

C13 C14

C11

C12

C15 O1

O2

C16

C17

O3

C19

C20 C21

C23

C24

C22

N3

N4

N5

N6

N

CA

C CB

O

CG

ND1

CD2

CE1

NE2

Tyr 188

Cys 32

Thr 296

Val 37

Thr 292

Leu 45

Ile 300

Gly 41

Asn 104

Pro 31

Ala 42

Ala 102

Ser 186

Leu 293

His 33

Lys 38

Glu 291

Olmesartan

His 297


We then decided to produce an AT2R1 model by compar-ative homology [40] with Bovine Rhodopsin[PDB:1L9H], but still could not produce a model whichwould dock the known ARBs, even after extensive energyminimization. Eventually we used the putative CCR2b,[PDB:1KP1] as the comparative model. Surprisingly,straight out of the 'Modeller' [41], all the ARBs dockedinto a pocket on the opposite side of the GPCR from thebinding pocket which had been located on CCR2b. The Kifor the ARBs ranged from 0.10 to 1.5 nmol, as detailed inTable 1.

It is interesting to note that although the comparativehomology between AT2R1 and Rhodopsin is only 17%(Table 2) the AT2R1 sequence is much closer to that ofCCR2b (Table 4). Our failure to produce a usable receptorby comparative homology with Bovine Rhodopsin wouldseem to caste doubt on its utility as a prototype for theClass A 7-transmembrane GPCR structures.

Figure 21 shows the primary residues involved in dockingthe ARBs, and a superimposition of the docked conforma-tions of Olmesartan and Losartan, demonstrating thehomogeneity of location of the imidazole group into thebinding pocket, even amongst ARBs with significant struc-tural differences.

The hydrophobic interactions between Olmesartan andour AT2R1 is shown in Figure 22. Olmesartan forms twohydrogen bonds, with GLY194 and LEU197, as does Losa-rtan (Figure 23). Candesartan binds to quite different res-

idues, in particular, making 6 hydrophobic contacts withILE193(Figure 24).

DiscussionModels provided to ease visualization of nuclear receptorsIt is evident from the lack of clarity in Figure 4 that it isextremely difficult to visualize ligand conformation in thebinding pockets of nuclear receptors using two dimen-sional media. For this reason we have provided, as anattached file, an archive of the receptor configurationsused in this study, in addition to the most significantbound ligand conformations. The models can be loadedinto, for example, the Python Molecular Viewer [35], and3D analysis performed.

This archive will also facilitate the testability of our results.

Does telmisartan selectively modulate PPARgamma?Benson, et.al. [20], presented the ARBs as suited to PPAR-gamma modulation. Their primary conclusion was thatTelmisartan's structure allowed it to exhibit selective mod-ulation, exhibiting in-vitro PPARgamma agonistic activityat low concentrations, changing to antagonistic activity athigher concentrations.

Figure 25 shows the key binding pocket for the agonistFarglitazar (GI262570) in the PPAR ligand bindingdomain. Figure 26, the LigPlot of this conformation,shows two key hydrogen bonds between Farglitazar's O1,HIS449 and TYR473, and two more between O2, SER289and HIS323.

Tsukahara, et.al. [52] recently studied a number of PPARagonists. They found that agonistic activity disappearswhen TYR473 is mutated, and noted the importance ofHIS323 and HIS449.

Figures 27 and 28 show the residues which contact PPAR-gamma when Irbesartan and Losartan are docked intotheir minimum energy conformations. Although Irbe-sartan hydrogen-bonds TYR473 and HIS449, Losartanonly contacts these residues, and forms its sole hydrogen-bond to ALA278. It would thus seem likely that Losartanis an effective PPAR antagonist. Irbesartan does not hydro-gen-bond to HIS323, a residue found critical to Rosiglita-zar's agonism [52], and probably is more likely anantagonist than agonist.

Figure 29 shows that Telmisartan does not form anyhydrogen bonds with the PPARgamma residues identifiedby Tsukahara, et.al., as critical to the agonistic activity ofRosglitazar. Any molecular mechanism which could resultin 'partial agonism' of PPARgamma by Telmisartan is stillto be elucidated

CCR2b-docked configurations for TAK779 and Olmesartan, individually and with superimpositionFigure 19CCR2b-docked configurations for TAK779 and Olm-esartan, individually and with superimposition. Note: Ligands depicted as "thick" and "thin" solely for visual clarity. Carbon atoms shown as grey, oxygen as red, nitrogen as blue, polar hydrogen as blue-white. Non-polar hydrogens not displayed.






Irbesartan docked into the CCR2b binding pocketFigure 20Irbesartan docked into the CCR2b binding pocket.

Key

Ligand bond

Non-ligand bond



contact


C1

C2

C6

C19

C3 C4

C5

C7

N1

C8 C9

N2 C10

C14

O1

C11 C12

C13

C15

C16

C17

C18

C20

C21 C22

C24

C25

C23

N3

N4

N5

N6

Tyr 188

Val 37

Cys 32

Leu 45

Thr 292

Thr 296

Pro 31

Asn 104

Ala 42

Leu 44

Gly 41

Ala 102

Ser 186

Ile 300

Lys 38

Glu 291

Irbesartan


We would note, however, that the extreme affinity whichTelmisartan exhibits for the ubiquitous VDR might wellalter expression of many hormones at concentrationslower than those at which Telmisartan begins to modulatePPARgamma. This may make it very difficult to evaluatecause and effect in the cascade of metabolic changeswhich will result from Telmisartan's blockade of the VDR.

Bovine and guinea pig AT2R1 for FDA in-vitro ARB studiesWhile modelling the ARBs docking into the AT2R1 recep-tor, we were struck by data in United States Food and DrugAdministration (US FDA) documents which did notexactly match our own observations.

For example, there are inconsistencies between our pre-dictions for the relative efficacies of Olmesartan, Cande-sartan and Losartan; and those of Figure 1.1.1.4 of FDANDA21-286 [2]. The NDA's in-vitro experiments, usingCavia porcellus, showed Olmesartan as having the highestARB efficacy, as we did, but found Candesartan close inefficacy to Olmesartan (1.2×) and Losartan to be lesseffective (3.4×). Our study found Losartan (Ki≈0.5 nmol)to be a better antagonist of AT2R1 than was Candesartan(Ki≈1.5 nmol).

The answer may well lie in sequence divergence betweenthe AT2R1 proteins from human, bovine, and guinea pigsources. The multiple sequence alignment showing differ-ences between AT2R1 from Homo sapiens, Cavia porcellusand Bos taurus is shown in Table 5.

Our model predicts that the primary residues involved indocking most of the ARBs are GLN15, GLY194, GLY196,THR198 and GLY203.

The binding pocket around GLN15 is conserved in allthree homologies.

However, in Bos taurus, the Isoleucine residue 193 ismutated to Valine. Candesartan has 6 hydrophobic con-tacts with ILE193, while Losartan and Olmesartan haveonly one. It is thus very likely that substitution of ILE193will differentially effect the degree of Candesartan's antag-onism of Bos taurus AT2R1 receptors, when comparedwith that of other ARBs, less dependent on contacts withILE193.

Additionally, there is a mutation in Leucine 205, structur-ally adjacent to GLY203. GLY203 has seven hydrophobiccontacts with Olmesartan, eight with Losartan, and sixwith Candesartan. In Cavia porcellus, this Glycine ismutated to Methionine.

The authors consequently believe that the FDA should re-examine the acceptability of Bos taurus and Caviaporcellustissues for demonstration of the efficacy of ARBs.

It was beyond the scope of this study to model AT2R1receptors for all three species used in the FDA in-vitrodata. This should form a topic for ongoing research.

Putative AT2R1 with (from left) Olmesartan, and Losartan docked, showing primary residues. Ligands are also shown superim-posedFigure 21Putative AT2R1 with (from left) Olmesartan, and Losartan docked, showing primary residues. Ligands are also shown superimposed. Note: Carbon atoms shown as grey, oxygen as red, nitrogen as blue, polar hydrogen as blue-white, and chlorine as green. Non-polar hydrogens not displayed. Residues displayed as 'CPK' charge spheres, ligands as 'ball and stick' models. Thick and thin ligand backbones displayed solely for visual clarity.




Olmesartan docked into the putative AT2R1 binding pocketFigure 22Olmesartan docked into the putative AT2R1 binding pocket. Note: The core structure of the hydrogen-bonded resi-dues is expanded to a 'ball-and-stick' format, so as to show the atoms involved in hydrogen bond formation.

Key

Ligand bond

Non-ligand bond



contact


2.65

2.88

C1

C2 C6

C18

C3

C4

C5

C7

N1

C8

C9

N2

C10

C13

C14

C11

C12

C15 O1

O2

C16

C17

O3

C19 C20

C21

C23

C24

C22

N3

N4 N5

N6

N

CA

C

O

N

CA

CB

C

CG

CD1

CD2

O

Gly 196

Asn 200

Gly 203

Leu 195

Thr 198

Leu 202

Gln 15

Lys 199

Gln 267

Glu 8

Thr 175

Phe 206

Thr 178

Ile 193

Olmesartan

Gly 194

Leu 197



Losartan docked into the putative AT2R1 binding pocketFigure 23Losartan docked into the putative AT2R1 binding pocket. Note: The core structure of the hydrogen-bonded residues is expanded to a 'ball-and-stick' format, so as to show the atoms involved in hydrogen bond formation.

Key

Ligand bond

Non-ligand bond



contact


2.962.49

C1

C2

C6

C7

C3

C4

C5

C16

N1

C8

C9 C10

C11

N2 C12

CL1

O1

C13

C14

C15

C17

C18

C19

C20

C22

C21

N3

N4

N5

N5

N CA

CB

C

CG

CD1

CD2

O

N

CA

C

O

Gly 196

Asn 200

Thr 198

Gly 203

Leu 195Leu 202

Lys 199

Gln 267

Thr 175

Phe 206

Gln 15

Thr 178

Ile 193

Losartan

Leu 197

Gly 194



Candesartan docked into the putative AT2R1 binding pocketFigure 24Candesartan docked into the putative AT2R1 binding pocket. Note: The core structure of the hydrogen-bonded res-idues is expanded to a 'ball-and-stick' format, so as to show the atoms involved in hydrogen bond formation.

Key

Ligand bond

Non-ligand bond



contact


3.17

C1 C2

C6

C7

C3

C4 C5

C18

N1

C8

C9

C10

C11

N2

O1

C12

C17

C14

C13

C15 C16

O2

O3

C19

C20 C21

C23

C24

C22

N3

N4

N5

N6

N

CA

CB

C

CG

CD OE1

NE2

O

Gly 196

Gly 203

Ile 270

Asp 263

Glu 8

Ile 193

Leu 195

Gly 194

Leu 202

Phe 171

Tyr 184

Lys 199

Phe 206

Asn 200

Asp 273

Thr 175

Thr 178

Leu 197

Thr 198

Candesartan

Gln 267


ConclusionThe FDA-approved prescribing information for Valsartanstates "Valsartan does not bind to or block other hormonereceptors or ion channels known to be important in cardi-ovascular regulation." This is an accurate statement of cur-rent knowledge about ARB in-vivo activity.

Yet this study found Valsartan (and the other ARBs) had aprofound affinity for the hormone receptor VDR, forPPARgamma and for CCR2b. Clearly, if our modellingdata sustains validation in the laboratory, clinical medi-cine will need to re-examine current concepts of howARBs function in-vivo. It is possible that ARBs maybecome useful as potent immunomodulatory agents inaddition to their current indication as cardiovasculardrugs. This study has shown how each ARB acts upon sev-eral key receptors of the immune system, and should serveas a solid basis for better understanding the anti-inflam-matory properties of this class of pharmaceutical.

MethodsHardware and molecular toolsTwo network servers were configured with Debian Linux.'AutoDock' [25,26,43,44] was kindly supplied by Scripps'and 'Modeller' by Salilab [41]. Ponder's 'Tinker' Toolsetwas downloaded from the cited location [39]. The Fortran

software sources for 'Modeller' and 'Tinker' were recom-piled with 64-bit Athlon-class optimizations applied, tosuit the 64-bit CPU. Each server had 1 Gigabyte of mem-ory, and a 160 Gigabyte hard disk. They were networked(using Samba [50]) to the primary Windows 2000 basedworkstation. The workstation also ran AutoDock (usingthe Cygwin executables), Python Molecular Viewer [35],and AutoDock Tools [34].

Optimization of the modelling software parametersAutodock uses a default grid size of 0.375 Angstroms. Thiswas changed to 0.2 Angstroms, noticeably improvingupon the Ki calculated with the coarser grid. However, thecomputing time with this more precise grid was increasedfour-fold. To ensure more reliable minima from Auto-Dock's Lamarckian genetic algorithm, the 'populationsize' parameter "ga_pop_size" was increased from 50 to100, and the number of energy calculations for each set,"ga_num_evals," was increased from 250,000 to1,000,000. One set of AutoDock grid maps was typicallygenerated for each receptor, and multiple ligands weredocked without changing the grid maps. Docking param-eter files were edited using the Linux ASCII text editor.

Energy minimization of structures with Ponder's 'mini-mize' and 'pss' [38] programs was effected using the'Amber99' parameter set [47].

Construction of ligand and receptor moleculesAkira Dobashi's 3D Pharmaceutical Structure Database atPharmis.org [48] (Tokyo University of Pharmacy and LifeSciences) was the primary source of ARB models. Olme-sartan had to be built with Ghemical [49], running on aLinux server. Receptor coordinates were taken from theRCSB Protein Databank (PDB), or generated using Mod-eller [41] (as detailed in the text).

LigPlot and HBPLUSMcDonald's HBPLUS software [56,55] takes, as input, thecomputed 3D ligand-receptor complex and produces atable of hydrogen bonds formed between the ligand andthe receptor. It also produces tables of non-bondinghydrophobic contacts between the ligand atoms andreceptor residues (default distance parameters were usedfor both bonds and contacts). Wallace and Laskowski'sLigPlot software [53,54] takes those tables and creates a2D representation of the bonds and contacts, iterativelyoptimizing the output against a set of user-specified plotparameters. For example, weight-parameters can beassigned to minimize areas where the plot of hydrophobicbonds becomes too dense, forcing LigPlot to iterativelymove the 2D positions of the residues so as to minimizethat clutter, and thus make the output more readable. Theoutput of Ligplot is PostScript, which was modified with

Farglitazar docked into the PPARgamma ligand binding pocket, showing the primary residues involved in hydrogen-bondingFigure 25Farglitazar docked into the PPARgamma ligand binding pocket, showing the primary residues involved in hydrogenbonding. Note: Ligand depicted with yellow backbone solely for visual clarity. Carbon atoms shown as grey, oxygen as red, nitrogen as blue, polar hydro-gen as blue-white. Non-polar hydrogens not displayed. Resi-dues displayed as 'CPK' charge spheres, ligand as 'ball and stick' model.




Farglitazar docked into the PPARgamma ligand binding domainFigure 26Farglitazar docked into the PPARgamma ligand binding domain. Note: The core structure of the hydrogen-bonded residues is expanded to a 'ball-and-stick' format, so as to show the atoms involved in hydrogen bond formation.

Key

Ligand bond

Non-ligand bond



contact


3.03

2.79

3.03

2.88 CG

CD1 CD2

CB

CE1 CE2

CZ

OH

CA

N

C

C1A

C1F

C1B

C1E C1G

C1C

C1D

O1G

C1H

C1I

C1M

C1J

C1L

C1K

O2

O1

C3A

C3B

C3C

N3H

C3D

C3G

O3F

C3E

C3I C3N

C3J C3M

C3K C3L

N

CA

C

CB

O

CG

ND1

CD2

CE1

NE2

N

CA

C

CB

O

OG

N

CA

C

CB

O

CG

ND1

CD2

CE1

NE2

N

CA

C

CB

O

CG

CD1

CD2

CE1

CE2

CZ OH

Cys 285

Arg 288

Gly 284

Gln 286

Ser 342

Phe 282

Phe 363

Tyr 327

Ile 281

Leu 465

Leu 469

Glu 291

Phe 360

Leu 453

Farglitazar

His 449

Ser 289

His 323

Tyr 473



Irbesartan docked into the PPARgamma ligand binding domainFigure 27Irbesartan docked into the PPARgamma ligand binding domain. Note: The core structure of the hydrogen-bonded residues is expanded to a 'ball-and-stick' format, so as to show the atoms involved in hydrogen bond formation.

Key

Ligand bond

Non-ligand bond



contact


2.49

3.03

C1

C2

C6

C19

C3 C4

C5

C7

N1

C8

C9

N2 C10

C14

O1

C11

C12

C13

C15

C16

C17

C18

C20

C21 C22

C24

C25

C23 N3

N4

N5

N6

N CA

C

CB

O

CG

CD1

CD2

CE1

CE2

CZ

OH

N

CA

C CB

O

CG

ND1 CD2

CE1 NE2

Cys 285

Phe 282

Leu 330

Gln 286

Leu 469

His 323

Arg 288

Ser 289

Met 364

Ile 326

Ile 341

Val 339

Leu 465

Leu 340

Leu 453

Irbesartan

Tyr 473

His 449



Losartan docked into the PPARgamma ligand binding domainFigure 28Losartan docked into the PPARgamma ligand binding domain. Note: The core structure of the hydrogen-bonded residues is expanded to a 'ball-and-stick' format, so as to show the atoms involved in hydrogen bond formation.

Key

Ligand bond

Non-ligand bond



contact


3.23

C1

C2

C6

C7

C3

C4

C5

C16

N1

C8

C9 C10

C11

N2 C12

CL1

O1

C13

C14

C15

C17

C18

C19

C20

C22

C21

N3

N4

N5

N6

N

CA

C

CB

O

Cys 285

Phe 360

Phe 282

Met 364

His 449

Leu 353

Phe 363

Ile 281

Tyr 473

Leu 356

Ser 289Leu 453

His 323

Tyr 327

Gly 361

Leu 469

Lys 354

Ile 456

Losartan

Ala 278



Telmisartan docked into the PPARgamma ligand binding domainFigure 29Telmisartan docked into the PPARgamma ligand binding domain.

Key

Ligand bond

Non-ligand bond



contact


C1

C2

C3

C26

C4 C6

C5

C7

N1

N2

C8

C9

C23

C10

C11 C12

C13 C15

C14

C16

C17

C18

C19

C22

C21

C20

O1

O2

C24

C25

N3

N4

C27

C28

C30 C29

C31

C33

C32

Cys 285

Phe 363

Ser 289

Arg 288

Phe 360

Phe 282

His 323

Ile 326

His 449

Ile 341

Leu 453

Met 348

Ile 456

Gln 286

Tyr 327

Val 339

Tyr 473

Gly 284

Ala 292

Leu 330

Leu 469

Telmisartan


Table 5: Multiple sequence alignment highlighting differences between AT2R1 from Homo sapiens, Cavia porcellus and Bos taurus.

sp|P30556|AGTR1_HUMANgi|8927995|sp|Q9WV26|AGTR1_CAVgi|27806329|ref|NP_776658.1|B.taurus





sp|P30556|AGTR1_HUMANgi|8927995|sp|Q9WV26|AGTR1_CAV gi|27806329|ref|NP_776658.1|B.taurus


sp|P30556|AGTR1_HUMANgi|8927995|sp|Q9WV26|AGTR1_CAV gi|27806329|ref|NP_776658.1|B.taurus

. . . . . . . . . . . . . . . . . . . . . . . . N . . . V . . . . . . . . . . . . . . . . . . . . . 5 0

. . . . . . . . . . . . . . . . . . . . . . . . S . . . V . . . . . . . . . . . . . . . . . . . . . 5 0

. . . . . . . . . . . . . . . . . . . . . . . . N . . . I . . . . . . . . . . . . . . . . . . . . . 5 0

************************ . *** : *********************

. . . . . . . . . . . . . . . . . . . . . . . . L . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 0

. . . . . . . . . . . . . . . . . . . . . . . . I . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 0

. . . . . . . . . . . . . . . . . . . . . . . . L . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 0

************************ : *************************

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I 1 5 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V 1 5 0

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I 1 5 0

************************************************* :

. . . . L . . . . . . . A I . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . . . . . 2 0 0

. . . .M . . . . . . . A V . . . . . . . . . . . . . . . . . . . . . . . . . . . . I . . . . . . . 2 0 0

. . . . L . . . . . . . T I . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . . . . . . . 2 0 0**** : ******* : : **************************** : *******

. . . . L . . . . . . . . . . . . . . . A . . . . . . . . . . . . . N . . . . . M . . . . . . . 2 5 0

. . . .M . . . . . . . . . . . . . . . A . . . . . . . . . . . . . N . . . . . M . . . . . . . 2 5 0

. . . . L . . . . . . . . . . . . . . . T . . . . . . . . . . . . . K . . . . . L . . . . . . . 2 5 0**** : ******* : : **************************** : *******

. . . I . . . . . . . L . . . . . . . I . R . . R . A . . . . . . . . . . . . I . . . . . . . . . . 3 0 0

. . . V . . . . . . . L . . . . . . . I . H . . K . S . . . . . . . . . . . . I . . . . . . . . . . 3 0 0

. . . V . . . . . . .M . . . . . . . L . R . . K . E . . . . . . . . . . . . L . . . . . . . . . . 3 0 0

*** : ******* : ******* : * : ** : * ************ : * * * * * * * * *

. . . . . . . . . . R . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . D . V S . . T . 3 5 0

. . . . . . . . . . K . . . . . . . . . . . . . . . . . T . . . . . . . . . . . . . D . V S . . A . 3 5 0

. . . . . . . . . . K . . . . . . . . . . . . . . . . . N . . . . . . . . . . . . . E . G N . . T . 3 5 0

********** : ***************** . ************* : * . ** : *

. . A P . F . . . 3 5 9

. . VQ . F . . . 3 5 9

. . A P . I . . . 3 5 9** . * : ***

a text editor to maximize font readability, and crop excesswhite space.

Competing interestsREL has no competing interests. TGM is designated asinventor on a US patent application titled "Treatment ofTh1 and autoimmune diseases effected with angiotensininhibition and antibiotics." No assistance has beenrequested or received by any of the authors from any phar-maceutical company, or other financially interestedentity. This study was entirely funded by the authors.

Authors' contributionsTGM conceived, designed, and carried out the molecularstudies, performed the 'Modeller' sequence alignments,configured the computer servers, the computer software,and drafted the manuscript. REL was responsible forreceptor phylogenies and Clustal alignments. FEM partic-ipated in the molecular model definition, coordinated theFDA and pharmacological issues, and helped to draft the

manuscript. All authors read and approved the final man-uscript.



Additional material

AcknowledgementsSpecial thanks to Meg Mangin, Belinda J Fenter, Barb Oberle, Lottie Stanley, and Karen Marshall.

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Additional File 1'ARB-immune-models.tar.gz' – Models of receptors and significant ligands. This is a Tar-Gzip archive which can by unpacked by using 'tar-xvzf', Winzipv8+ or 'Mac Stuffit'. There are five directories within it, con-taining a total of 35 files, 1.54 Meg when unpacked, 360 K when com-pressed: • 'AT2R1' contains the receptor model described in this paper, plus the docked conformation of each ARB, corresponding to the Ki values in Table 1. • 'CCR2b' contains the receptor model we derived from 1KP1, together with TAK779 and each of the ARBs in their docked conforma-tion. • 'PPAR' contains the receptor model we derived from PDB:1FM9 together with the GI262570 ligand from PDB:1FM9 and each of the ARBs which have low Ki values when docked with the receptor. • 'VDR_from_1DB1" contains the VDR model we derived from PDB:1DB1 along with the docked conformations of the ARBs, 1,25-D (from PDB:1DB1) and TX522 (from PDB:1TXI). • 'VDR_from_1TXI' contains the VDR model we derived from PDB:1TXI along with the docked conformation of TX522, 1,25-D and Telmisartan.Click here for file[http://www.biomedcentral.com/content/supplementary/1742-4682-3-1-S1.gz]








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http://www.bioinformatics.org/ghemical/

http://www.bioinformatics.org/ghemical/



http://www.biochem.ucl.ac.uk/bsm/ligplot/ligplot.html

http://www.biochem.ucl.ac.uk/bsm/ligplot/ligplot.html



http://www.biochem.ucl.ac.uk/bsm/hbplus/home.html

http://www.biochem.ucl.ac.uk/bsm/hbplus/home.html





http://www.biomedcentral.com/info/publishing_adv.asp


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